Reactive oxygen species (ROS) generated after cerebral ischemia and reperfusion activate both caspase-dependent and caspase-independent pathways leading to delayed neuronal death. This project will use a combination of in vivo and in vitro models of ischemia, viral-mediated gene transfer, and knockout models to address the central role of oxidative stress and ROS in initiating key apoptotic pathways. It will also explore the temporal dynamics and interactions between those pathways, and determine whether hypothermia alters those dynamics and interactions, and thus enhances neuroprotection. Additionally, preservation of neuronal function conferred by gene transfer therapy will be examined. First, we will explore the role of reactive oxygen species (ROS) in activating both caspase-dependent and -independent apoptotic events, assess whether and how they are altered by over-expression of anti-oxidant genes, whether such over-expression protects ischemia-vulnerable SOD2 knockout mice, and explore effects of deltaPKC on ROS and apoptosis post-ischemia. Second, we propose to examine the interactions between various mediators of apoptotic pathways after cerebral ischemia. We will investigate the effects of gene therapy using over-expression of the caspase inhibitors p35 and crmA, as well as pharmacologic caspase antagonists, and examine their effects on interactions between AIF- and caspase-dependent apoptotic pathways after ischemia. Finally, we will investigate whether post-ischemic hypothermia prolongs the temporal therapeutic window for gene therapy against global cerebral ischemia, and whether gene therapy with or without hypothermia spares neuronal function. Specifically, we will determine whether hypothermia blocks or delays ROS activity and apoptotic mediators, prolongs the time window for protection by over-expression of GPX, catalase, or other anti-apoptotic proteins, and permits gene therapy to spare neuronal function following global ischemia. We believe that by combining well-established in vivo and in vitro models of stroke with gene transfer and transgenic technology, we can apply unique and novel approaches to elucidate ROS-related mechanisms of neuronal death, survival, and function.